US20030166005A1 - Test system for determining the activity of cyclo-nucleotide-dependent protein kinases and vasp phosphatases - Google Patents

Test system for determining the activity of cyclo-nucleotide-dependent protein kinases and vasp phosphatases Download PDF

Info

Publication number
US20030166005A1
US20030166005A1 US10/311,093 US31109303A US2003166005A1 US 20030166005 A1 US20030166005 A1 US 20030166005A1 US 31109303 A US31109303 A US 31109303A US 2003166005 A1 US2003166005 A1 US 2003166005A1
Authority
US
United States
Prior art keywords
vasp
cnpk
test system
substrate
phosphatase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/311,093
Other languages
English (en)
Inventor
Peter Druckes
Thomas Jarchau
Ulrich Walter
Benjamin Bader
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Verinos Operations GmbH
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Assigned to VASOPHARM BIOTECH GMBH reassignment VASOPHARM BIOTECH GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BADER, BENJAMIN, DRUCKES, PETER, JARCHAU, THOMAS, WALTER, ULRICH
Publication of US20030166005A1 publication Critical patent/US20030166005A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates to a test system for detecting the activity of cyclonucleotide-dependent protein kinases or of phosphatases which dephosphorylate the vasodilator-stimulated phosphoprotein (“VASP”), to methods for preparing it and to its use in (high-throughput) screening methods.
  • VASP vasodilator-stimulated phosphoprotein
  • Cyclonucleotide-dependent protein kinases (“cNPKs”) their substrates (“cNPKSs”), and also the corresponding protein phosphatases, are part of important physiologically, pathophysiologically and pharmacologically relevant cellular signal pathways (cf. references 1, 2 and 3).
  • the cNPKs include cAMP-dependent protein kinases (“cAKs”) and cGMP-dependent protein kinases (“cGKs”) .
  • cGK is, in particular, an important mediator of cGMP-mediated signal transmission.
  • GC-A, GC-B and GC-C membrane-located guanylyl cyclases
  • ANP, BNP and CNP natriuretic peptides
  • enterotoxin/guanylin enterotoxin/guanylin
  • GC-S soluble guanylyl cyclase
  • cGMP smooth muscle cells
  • cGKs are known to be homodimers, of which a soluble form (76 kDa monomer; cGK I) and a membrane-located form (86 kDa monomer; cGK II) can be distinguished.
  • the anchoring of cGK II to the membrane is mediated by way of N-terminal myristoylation (cf. references 1-3).
  • Two isoforms of cGK I exist, i.e. cGK I ⁇ and cGK I ⁇ .
  • cGK I which is universally described as the soluble form, is predominantly present in blood platelets in particulate form and partially present in particulate form in smooth muscle cells.
  • cGK I A particularly high expression of cGK I is found in human blood platelets and in smooth muscle cells. Activation of cGK-I in these cells leads to the effects which have already been described above, such as a lowering of the intracellular level of Ca(2+), inhibition of blood platelets and contraction of smooth muscle cells.
  • the known substrates of cGK include, for example, the inositol 1,4,5-triphosphate receptor (IP3R) and VASP, and also p25, cystic fibrosis transmembrane regulator (CFTR) and 6-pyruvoyltetra-hydropterin synthase (PTPS).
  • IP3R inositol 1,4,5-triphosphate receptor
  • VASP VASP
  • CFTR cystic fibrosis transmembrane regulator
  • PTPS 6-pyruvoyltetra-hydropterin synthase
  • Disturbances of the ANF/NO/cGMP signal pathway are known for the different forms of atherosclerosis and hypertension and what is termed “endothelial dysfunction”, i.e. a disturbance of endothelium-mediated vasodilatation, is an early sign of vascular diseases associated with atherosclerosis, hypertension and diabetes (cf. references 6 and 7).
  • Endothelial dysfunction i.e. a disturbance of endothelium-mediated vasodilatation
  • Current findings provide evidence that the activation and/or expression of soluble guanylyl cyclase, in particular, is pathologically decreased in these vascular diseases (cf. references 8-10). It follows conclusively from this that compounds/substances which are able to replace the NO/cGMP signal pathway are of very great therapeutic importance.
  • cNPK assays are customarily carried out by incorporating radioactive phosphorus isotopes ( 32 P or 33 P) into peptide substrates and subsequently separating these reaction products from the starting compounds, for example by immobilizing them on a carrier substance (reference 16), or by separating the reaction products by means of gel electrophoresis, and carrying out a radiochemical or an immunochemical detection following protein transfer to membranes when product-specific antibodies are available (as, for example, in a Western blot, WO 99/24,473).
  • test system synthetic: assay or diagnostic agent
  • test system synthetic: assay or diagnostic agent
  • HTS high-throughput screening
  • test system in a simple and effective manner, to analyze a large number of (test) compounds from chemical or natural substance libraries for their effect on the activity of a VASP phosphatase [so-called high-throughput screening (abbreviated to HTS)].
  • HTS high-throughput screening
  • One part of the subject matter of the present invention is therefore an HTS-suitable test system for detecting the activity of a cNPK, comprising
  • HTS-suitable test system for detecting the activity of the VASP phosphatase comprising
  • test system As a gel-free test system, the test system according to the invention makes it possible to carry out a large number of individual quantitative determinations and, by means of dispensing with gel-electrophoretic separations steps, fulfills particular requirements with regard to speed, sample size and consumption of materials. Furthermore, the test system ensures reproducibility, robustness, solvent tolerance and automation.
  • test system makes it possible to search for one or more, identical or different test compounds.
  • the cNPK used is preferably a cGK or cAK or a functional variant which possesses the activity of cNPK.
  • Such protein kinases can be used in purified form or as a crude extract, in particular as a constituent in protein mixtures derived from homogenates of biological origin, preferably of human origin.
  • VASP phosphatase used can likewise be a functional variant which possesses the activity of a VASP phosphatase.
  • VASP phosphatases can be used in purified form or as a crude extract, in particular as a constituent in protein mixtures derived from homogenates of biological origin, preferably of human origin.
  • VASP as depicted in SEQ ID No. 1 (described in C. Haffner et al. in EMBO J., 14(1), 19-27 (1995)), containing preferably operable phosphorylation sites, namely serine-157, serine-239 and threonine-278, is very particularly preferred for use as a suitable cNPK substrate in accordance with feature (b).
  • cNPKs can in principle phosphorylate all three residues, however, the cAMP-dependent protein kinase prefers serine 157 as the main phosphorylation site, while the cGMP-dependent protein kinase prefers serine-139.
  • Threonine-278 is phosphorylated with comparable specificities by both kinases.
  • Phosphorylated VASP corresponding to SEQ ID No. 1, which is phosphorylated at the preferably operable phosphorylation sites, specifically serine-157, serine-239 and threonine-278, is very particularly preferred for use as a suitable VASP phosphatase substrate in accordance with feature (b).
  • a cNPK substrate or of a VASP phosphatase substrate those peptide or polypeptide variants, or peptoids, which preferably contain VASP amino acid sequences which are present in the environment of said phosphorylation sites or dephosphorylation sites, with particular preference being given, but not conclusively, to the pentamers containing amino acids 155-159 and/or 237-241 and/or 276-280 as depicted in SEQ ID No. 1, or preference being given to the decamers containing amino acids 152-161 and/or 234-243 and/or 273-282 from SEQ ID No. 1, or other suitable constituent sequences (e.g., cf. in FIG. 1).
  • the monoclonal antibody 16C2 which is selected and obtainable from the hybridoma cell line DSM ACC2330 (WO 99/24473).
  • This antibody, or a functional variant thereof specifically detects a phosphorylation event; very particularly preferably it recognizes an epitope which contains the phosphorylated serine-239 of the VASP sequence.
  • a functional variant relates, in particular, to those antibodies having a congruence of preferably 90-99% or 70-90% with the amino acid sequence of 16C2 with the homologous function of quantitatively determining a reaction product within the meaning of feature (d) or (h), respectively.
  • test system can be operated heterogeneously (e.g. after immobilizing the reaction products) it is preferably operated homogeneously.
  • immunological detection within the meaning of features (d) and (h), respectively can be effected using methods which are known to the skilled person, such as a coupled enzyme reaction (alkaline phosphatase or luciferase) or general sandwich technologies (for example ELISA) and also fluorimetric methods (fluorescence, time-resolved fluorescence and fluorescence resonant energy transfer (FRET)).
  • coupled enzyme reaction alkaline phosphatase or luciferase
  • sandwich technologies for example ELISA
  • fluorimetric methods fluorescence, time-resolved fluorescence and fluorescence resonant energy transfer (FRET)
  • test system according to the invention can be automated as a homogeneous HTS and is therefore suitable for 96-, 384- and 1536-well plates and more.
  • supplementary agents such as buffer solutions, stabilizers and/or energy equivalents, in particular ATP, are preferably used for carrying out the investigations.
  • the present invention also relates to a method for preparing a test system in which at least one compound to be investigated and at least one composition, containing cNPK, ATP and cNPK substrate, where appropriate a phosphorylation reaction stopper, at least one suitable detection system, such as an antibody and an immunological detection means, and, where appropriate, further supplementary agents, are combined.
  • the present invention also relates to a method for preparing a test system in which at least one compound to be investigated, at least one composition containing VASP phosphatase and at least one detection system which is suitable for quantifying the dephosphorylation of a VASP phosphatase substrate, are combined.
  • Another part of the subject matter of the present invention is an HTS-suitable method for finding one or more active compounds which modulate the activity of the cNPKs, comprising the steps of:
  • Another part of the subject matter of the present invention is an HTS-suitable method for finding one or more active compounds which modulate the activity of the VASP phosphatase, comprising the steps of:
  • the method for finding a chemical compound is carried out in a microtiter plate.
  • the microtiter plate can contain differing numbers of wells.
  • the plate can contain 96, 384, 768, 1536, 3072, or more, wells. Individual components of the method according to the invention which are preferred have already been described in detail above.
  • the active (test) compound can be a pharmaceutically active compound, specifically having the function of a modulator, such as a (hyper)activator/or (total) inhibitor, of the activity of a cNPK, or be a natural product in the widest possible sense, in particular a crude extract or a component which is contained therein.
  • a modulator such as a (hyper)activator/or (total) inhibitor
  • the substance to be investigated is generally a naturally occurring, a naturally occurring and chemically modified and/or synthetic substance.
  • Such a compound is very particularly preferably suitable which, while circumventing the NO/cGMP signal pathway, modulates a cNPK directly—activates or inhibits; optionally, selectively modulates—activates or inhibits,.
  • the invention also relates to a method in which the pharmaceutically active compound modulates a cNPK signal pathway.
  • Another part of the subject matter of the present invention is therefore a method for diagnosing by directly, and in a gel electrophoresis-independent manner, determining the cNPK activity in samples, comprising the steps of:
  • Another part of the subject matter of the invention is a method for diagnosing by directly, and in a gel electrophoresis-independent manner, determining the VASP phosphatase activity in samples, comprising the steps of:
  • VASP phosphatase determining the activity of the VASP phosphatase, in particular in blood extracts, cell extracts or tissue extracts or in samples which are in the form of permeabilized and/or intact cells.
  • the diseases which are to be diagnosed are preferably angiocardiopathies and diseases which are associated with vascular damage, for example hypertension, thrombosis and the syndrome of endothelial dysfunction, for example in arteriosclerosis, diabetes, vasculites and diseases of hematopoietic cells, such as acute leukemias, myeloproliferative diseases and myelodysplasias (cf. reference 17).
  • FIG. 1 shows a preferred embodiment of the test system according to the invention.
  • the assay of cGK activity is based on observing the in-vitro phosphorylation of a biotinylated substrate peptide by activated cGK (compare FIG. 1A).
  • the VASP substrate peptide consists of a part of the amino acid sequence of human VASP (vasodilator-stimulated phosphoprotein), which is a natural cGK substrate protein.
  • VASP vasodilator-stimulated phosphoprotein
  • FIG. 1B time-resolved fluorescence resonance energy transfer
  • a europium-labeled anti-mouse IgG second antibody which can specifically bind to the anti-pSer239-VASP (mAb 16C2).
  • SA-APC Fluorescence-labeled streptavidin
  • FIG. 2 shows the kinetics of the phosphorylation of the biotinylated substrate peptide by the cGK.
  • the ordinate in FIG. 2 shows the FRET signal measured in cps.
  • the VASP substrate peptide is only phosphorylated in the presence of the cGK and its activator cGMP (tube A). No change in signal was observed without cGMP and with cGK (tube B) and without cGK and with cGMP (tube C).
  • FIG. 3 shows the activation of the cGK with the natural activator cGMP and with a cGMP-analogous molecule (8-pCPT-cGMP), as measured using the described test system.
  • the enzyme activity (in cps/min) is plotted against the activator concentration (in mol/l).
  • FIG. 4 shows the surprising finding that the test system according to the invention can be used to observe, and quantitatively measure, the enzyme activity of the cGK contained in thrombocyte extracts even in this complex biological mixture of protein.
  • FIG. 4A shows no difference between the phosphorylation kinetics without ( ⁇ ) and with ( ⁇ ) the specific cAK inhibitor PKI.
  • FIG. 4B depicts the influence of 100 nmol of PKI/l on the phosphorylation of the VASP substrate peptide by purified cGK (0.3 nmol/l) without ( ⁇ ) and with ( ⁇ ) PKI, and also the influence of PKI on purified cAK (catalytic subunit, 3 nmol/l) without ( ⁇ ) and with ( ⁇ ) PKI.
  • a synthetic phosphopeptide (phosphorylated version of the VASP substrate peptide) was used as the “reaction product” and was quantified using a detection mix.
  • reaction product phosphorylated version of the VASP substrate peptide
  • different mixtures of unphosphorylated peptide and phosphopeptide were prepared. When so doing, the total concentration of peptide was kept constant at 500 nmol/l.
  • the time-resolved FRET signal when exciting the europium fluorescence at 340 nm and measuring the emission of the APC fluorescence at 660 nm, was measured, after incubating at 20° C. for 1 hour, using a PerkinElmer Wallac fluorescence-measuring instrument (Victor 2 ).
  • the following Table shows a virtually linear increase between 0 and 100 nmol/l of phosphopeptide. At 160 nmol/l of phosphopeptide, the signal cannot be increased any further.
  • the signal/background ratio as defined by the quotient of the maximum signal and signal without phosphopeptide, was about 5.
  • an optimum signal/background ratio was obtained when 20 ⁇ l of a solution containing 500 nmol of VASP substrate peptide/l were mixed with 40 ⁇ l of a detection mix which contained the abovementioned concentrations of the detection reagent. This protocol was used in all the subsequent experiments.
  • the activity of an enzyme reaction which is carried out in a volume of 100 ⁇ l and in which a signal of 9872 cps is measured within 2 minutes corresponds to an activity which transforms 2 pmol of substrate in 2 minutes, corresponding to an enzyme activity of 1 ⁇ 10 ⁇ 6 U.
  • Tube A additionally contained 5 ⁇ mol/l of cGMP and 25 ng/ml of purified gGK.
  • Tube B additionally contained 25 ng/ml of purified gGK.
  • Tube C additionally contained 5 ⁇ mol/l of cGMP.
  • the kinase reaction was started with 50 ⁇ mol of ATP/l, with subsequent incubation being at 30° C. All the above-mentioned concentrations are to be understood as meaning final concentrations in a volume of 500 ⁇ l.
  • 50 ⁇ l were removed from the tubes and mixed with 50 ⁇ l of a 30 mmol/l solution of EDTA.
  • the phosphorylated substrate peptide was detected, as described in Example 1, in the wells of a 384-well microtest plate.
  • VASP substrate peptide is only phosphorylated in the presence of cGK and its activator cGMP (tube A). Without cGMP and with cGK (tube B), as well as without cGK and with cGMP (tube C), no change in signal was observed.
  • the test system according to the invention is suitable for high-throughput screening.
  • Microtest plates in the 96-well, 384-well and even 1536-well format can be processed using commercially available pipetting robots (e.g. the Biomek Systems from Beckman or the Genesis Workstation from Tecan).
  • the cGK assay could be implemented in the following way, on such a robot system in the 384-well format, for screening for activators of cGK. The following are pipetted in sequentially:
  • test system can be used to analyze the dose-dependent activation of cGK by the natural activator cGMP and a molecule which is analogous to cGMP (8-pCPT-cGMP).
  • 8 kinase reactions as described in Example 3, total volume 500 ⁇ l were carried out, with these reactions containing the following components:
  • test system is suitable for specifically measuring the activity of cGK in tissue extracts or thrombocyte lysates.
  • Thrombocytes were prepared using a known method (cf. reference 15). After a soluble thrombocyte extract had been prepared, it was tested without activator and with 8-pCPT-cGMP (0.5 ⁇ mol/l) as described in Example 3. In addition to 8-pCPT-cGMP, a third reaction tube also contained 100 nmol/l of PKI. This inhibitor specifically inactivates cAMP-dependent protein kinase (cAK), which is also present in thrombocytes.
  • FIG. 4A shows no difference between the phosphorylation kinetics without and with PKI.
  • FIG. 4A shows no difference between the phosphorylation kinetics without and with PKI.
  • 4B depicts the influence of 100 nmol of PKI/l on the phosphorylation of the VASP substrate peptide by purified cGK (0.3 nmol/l) and purified cAK (catalytic subunit, 3 nmol/l) .
  • concentration of PKI which is employed is sufficient to inhibit the cAK completely.
  • the activity of the cGK is unaffected.
  • FIGS. 4A and 4B therefore together show that the test system according to the invention can measure the cGK activity in complex samples, such as thrombocyte lysates, very specifically. Any cross reactivity due to cAK can be ruled out. It was likewise possible to measure cGK activity in tissue homogenates prepared from rat organs.
  • cAMP cycloadenosine-3′, 5′-monophosphate
  • APC allophycocyanin
  • ATP adenosine-5′-triphosphate
  • BSA bovine serum albumin
  • cAK cAMP-dependent protein kinase
  • cGK cGMP-dependent protein kinases
  • cGMP cycloguanosine-3′, 5′-monophosphate
  • cNPKS substrate for cyclonucleotide-dependent protein kinases
  • cNPK cyclonucleotide-dependent protein kinases
  • LANCE lanthanide chelate excitation technology
  • mAb monoclonal antibody
  • PKI protein kinase A-specific inhibitor
  • TR-FRET time-resolved fluorescence resonance energy transfer
  • VASP vasodilator-stimulated phosphoprotein

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US10/311,093 2000-06-14 2001-06-12 Test system for determining the activity of cyclo-nucleotide-dependent protein kinases and vasp phosphatases Abandoned US20030166005A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10029210.0 2000-06-14
DE10029210A DE10029210A1 (de) 2000-06-14 2000-06-14 Testsystem sowie dessen Verwendung

Publications (1)

Publication Number Publication Date
US20030166005A1 true US20030166005A1 (en) 2003-09-04

Family

ID=7645644

Family Applications (1)

Application Number Title Priority Date Filing Date
US10/311,093 Abandoned US20030166005A1 (en) 2000-06-14 2001-06-12 Test system for determining the activity of cyclo-nucleotide-dependent protein kinases and vasp phosphatases

Country Status (5)

Country Link
US (1) US20030166005A1 (fr)
EP (1) EP1294926A2 (fr)
AU (1) AU2001263956A1 (fr)
DE (1) DE10029210A1 (fr)
WO (1) WO2001096594A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107709996A (zh) * 2015-05-08 2018-02-16 分析科学莱布尼茨研究所Isas协会 鉴定用于凝血障碍的诊断和风险分层的标记蛋白的方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10058596A1 (de) * 2000-11-25 2002-06-06 Aventis Pharma Gmbh Verfahren zum Screening von chemischen Verbindungen zur Modulierung der Wechselwirkung einer EVH1-Domäne oder eines Proteins mit einer EVH1-Domäne mit einer EVH1-Bindedomäne oder einem Protein mit einer EVH1-Bindedomäne sowie ein Verfahren zum Nachweis besagter Wechselwirkung
CA2445420A1 (fr) * 2003-07-29 2005-01-29 Invitrogen Corporation Tests de kinase et de phosphatase
US7727752B2 (en) 2003-07-29 2010-06-01 Life Technologies Corporation Kinase and phosphatase assays
US20070264678A1 (en) 2005-10-28 2007-11-15 Invitrogen Corporation Kinase and ubiquination assays

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6346406B1 (en) * 1997-08-20 2002-02-12 University Of Medicine & Dentistry Of New Jersey Elongation factor-2 kinase (EF-2 kinase), and methods of use therefor
WO1999024473A1 (fr) * 1997-11-07 1999-05-20 Aventis Pharma Deutschland Gmbh Anticorps contre une phosphoproteine phosphorylee stimulee par un vasodilatateur (vasp), cellules d'hybridome permettant leur preparation et utilisation desdits anticorps

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107709996A (zh) * 2015-05-08 2018-02-16 分析科学莱布尼茨研究所Isas协会 鉴定用于凝血障碍的诊断和风险分层的标记蛋白的方法

Also Published As

Publication number Publication date
WO2001096594A2 (fr) 2001-12-20
EP1294926A2 (fr) 2003-03-26
DE10029210A1 (de) 2002-01-31
WO2001096594A3 (fr) 2002-05-16
AU2001263956A1 (en) 2001-12-24

Similar Documents

Publication Publication Date Title
US5759787A (en) Kinase assay
US20070015231A1 (en) Assay for protein tyrosine phosphatases
EP1261621B1 (fr) Bioanalyses realisees au cours de la phosphorylation
JPH10511260A (ja) 個々のタンパクキナーゼ活性の定量法
US20030166005A1 (en) Test system for determining the activity of cyclo-nucleotide-dependent protein kinases and vasp phosphatases
US6127136A (en) Detection of dioxin-like compounds by detection of transformed Ah receptor/ARNT complex
EP0938586A1 (fr) Mesure de l'activite de la kinase par polarisation de fluorescence
EP0373908B1 (fr) Essai de fibrinolyse
Conroy et al. A rapid and sensitive fluorescence assay for angiotensin-converting enzyme
Cummings et al. Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: application to human immunodeficiency viral protease
Schurter et al. Analysis of isoaspartate in peptides and proteins without the use of radioisotopes
US20020192719A1 (en) Homogeneous assay methods
Maeda New label enzymes for bioluminescent enzyme immunoassay
Takeda et al. High-throughput kinase assay based on surface plasmon resonance suitable for native protein substrates
US20010004522A1 (en) Kinase activity measurement using fluorescence polarization
WO2002014543A2 (fr) Procede de detection d'activite enzymatique dans un dosage immunologique
EP0943919B1 (fr) Une surface d'essai qui permet une étape de libération d'analyte
Nyalala et al. Indirect enzyme-linked method for determining biotin in human serum
Landon et al. The measurement of enzymes by radioimmunoassay: A review
US20070026475A1 (en) Kinase and phosphatase activity measurement using fluorescent polarization
US4778755A (en) Immunoassay method
US20030027236A1 (en) Kinase activity measurement using flourescence polarization
US20040082021A1 (en) Method for assaying compounds or agents for ability to decrease the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase
Sironi et al. Tubulin carboxypeptidase assay based on the action of the enzyme on [14C] tyrosinated tubulin bound to nitrocellulose membrane
Ayers et al. Theophylline Assay on Kodak Ektachem DTSC—Performance and Interference by Structurally-Related Compounds and Salicylate

Legal Events

Date Code Title Description
AS Assignment

Owner name: VASOPHARM BIOTECH GMBH, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DRUCKES, PETER;JARCHAU, THOMAS;WALTER, ULRICH;AND OTHERS;REEL/FRAME:013908/0731;SIGNING DATES FROM 20030305 TO 20030311

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION